Project summary A long-standing goal in neuroscience is to unravel complex behavior of healthy and diseased brain by analyzing the structure and dynamics of neural circuitry with single action potential resolution. While many voltage-sensitive indicators have been developed for direct imaging of cellular membrane potentials, realization of their in vivo potential is still compromised by toxicity, time resolution and signal weakness arising from nonspecific background labeling, low quantum yields, limited dynamic range and signal dampening from increased cellular capacitance. Advances in de novo synthetic redox protein design can now be directed to overcome each of these limitations. We propose to exploit the adaptability of de novo protein design and well-understood rules of intraprotein electron tunneling to gain leveraged microsecond voltage sensitivity, sufficiently fast to resolve the entire action potential waveforms in neurons. Optical detection of the proposed transmembrane electron transfer (ET) relay will be achieved via additional electron or energy transfer with fused genetically encoded near-infrared fluorescent proteins (FPs). We expect these voltage sensors to be much faster and more tunable than current GEVIs that are based on voltage-dependent protein structural rearrangements with fundamental kinetic limit of ~0.5 millisecond. When developed, these sensors will greatly advance optical imaging of neural activity, thereby accelerating progress toward understanding how brain activity governs human behavior, cognition, and abnormal pathologies.